Phytoestrogens occur in a variety of plants, including vegetable protein materials such as soybeans. Phytoestrogens are defined as plant substances that are structurally and functionally similar to the gonadal steroid 17 .beta.-estradiol or that produce estrogenic effects. There are three main groups of nonsteroidial dietary estrogens which are 1) the isoflavones, 2) the coumestans and 3) the mycoestrogens (fungal). The structural similarity between these substances and the endogenous mammalian estrogens have been studied. A review of phytoestrogens and their effects in mammals is reported by Kaldas and Hughes in an article entitled, "Reproductive and General Metabolic Effects of Phytoestrogens in Mammals", Reproductive Toxicology, Vol. 3, pp. 81-89, 1989. The teachings of this article are herein incorporated by reference. As used in this specification and the appended claims, the term "isoflavones" is equivalent to the term "phytoestrogens" as that term is defined in the Kaldas et al. article. Representative of the isoflavones that are reduced in plant proteins in accordance with the present invention are daidzein, daidzin, genistein and genistin.
Isoflavones are produced by numerous leguminosoe and grasses, including many plants commonly consumed by man and livestock. Soy isoflavones include compounds such as daidzin, genistin, daidzein and genistein. A general structural formula for these compounds is:
______________________________________ #STR1## Compound R R.sub.1 ______________________________________ daidzein H H genistein H OH daidzin G H genistin G OH ______________________________________ wherein G = glucosyl
It has recently been recognized that isoflavones contained in vegetable proteins may have a detrimental impact upon the mammals that consume the vegetable protein. See Kaldas et al., supra. The concentration of isoflavones in plant protein isolates or concentrates such as soy protein isolates, can be as high as 3,000 .mu.g/g of protein. Isoflavones also provide the bitter or "beany" taste to vegetable proteins (see Ewan et al., infra), may reduce the bioavailability of essential minerals and may influence the nutritional value of proteins (see Kaldas et al., supra).
In contrast to the above recited negative aspects of isoflavones, it has been suggested that isoflavones may inhibit the growth of human cancer cells. Estrogens have two opposing effects on cancer, depending on dosage. Large doses inhibit breast cancer tumor development, while small doses seem to promote tumor growth. This duality extends to phytoestrogens or isoflavones. Isoflavones may stimulate or inhibit tumor growth. Setchell K D R, and Welch, M B J. Chrom. 386 (1987) 315-323; "Naturally Occurring Non-Steroidial Estrogens of Dietary Origin." In McLachlan J. A., ed. "Estrogens in the Environment", New York: Elsevier Press; 1985: 69-85 and Setchell, et al., "Nonsteroidial Estrogens of Dietary Origin: Possible Roles in Hormone-Dependent Disease", Am J. Clin. Nutr. 1984; 40: 569-578. One mechanism by which isoflavones may manifest their anti-tumor effect is blockage of estrogen receptors and uncoupling of receptor mediated response. Thus, the ability of endogenous estrogens to support tumor growth would be reduced. There is also indirect, demographic support for an isoflavone mediated reduction in cancers of hormone responsive tissues from the observation that women in countries consuming vegetarian diets have a lower incidence of breast cancer compared to meat-eating countries. Adlercreutrz et al., "Determination of Urinary Lignans and Phytoestrogen Metabolites, Potential Antiestrogens and Anticarcinogens, in Urine of Women on Various Habitual Diets" Steroid Biochem. 1986; 25: 791-797. Isoflavones may also have antiviral and fungicidal properties. Isoflavones have also been implicated in the reduction of serum cholesterol levels in humans, positive immunological effects and activity as an antioxidant. A final beneficial isoflavone effect is alleviation of vasomotor symptoms in menopausal women. Historically, the Chinese have used herbal medicine to treat "hot flashes". Thus, a process that facilely isolates and concentrates the isoflavones from plant material would be of value to the scientific community and the pharmaceutical industry.
Ion-exchange technology has been known for a great number of years. Ion-exchange resins are typically synthetic, insoluble, cross-linked polymers carrying acidic or basic side groups. They have high exchange capacities and can be used for an almost unlimited number of reactions. Ion-exchange resins are used in water-treatment, extraction, separation, analysis and catalysis.
Ion-exchange resins have an extended, open molecular framework that includes electrically charged ionic groups. A cation exchanger exchanges positive ions and therefore has negative ions built into its framework. An anion exchanger has positive ions in its framework. The ions of the lattice are called the fixed ions; the smaller ions of opposite charge that can change places with ions in the solution are called counterions.
Common problems encountered with ion exchange processes conducted on proteins include poor protein recovery (i.e., protein adhered to the resin) and inability of the protein slurry to pass through the resin bed resulting in a high pressure drop across the resin bed. The process which is disclosed herein fulfills the need in the pharmaceutical industry for a source of isoflavones that is economical and can be used on a commercial scale.
U.S. Pat. No. 5,352,384 to Shen discloses a process to produce an isoflavone enriched vegetable protein fiber. This patent discloses the use of a glucosidase to convert the glucone isoflavones (i.e., daidzen) in a protein slurry to the aglucone isoflavones. The fiber fraction is then recovered from the slurry by centrifugation to provide an aglucone enriched fiber.
In an article published in the Journal of Food Science, Vol. 47 (1982), pp. 933-940, by J. How and C. Morr entitled "Removal of Phenolic Compounds from Soy Protein Extracts Using Activated Carbon", they report subjecting soy protein extracts to activated carbon and ion exchange process treatments to remove phenolic compounds that have been reported as being responsible for adverse color and flavor characteristics of soy protein products. Protein extracts were subjected to a two stage, sequential ion exchange treatment prior to protein precipitation. The protein extract was pumped "down-flow" through a cation exchange column in the Na+ form and then an anion exchanger in the OH.sup.- /Cl.sup.- form to remove polyvalent anions including phenolic acids, phytate and others.
U.S. Pat. No. 5,248,804 to Nardelli et al. discloses a process for the removal of phytate from plant protein using ion-exchange resins. The process uses a macroporous anion exchange resin (weak base or strong base) which has been conditioned by 1) conversion to the hydroxide form; 2) conversion to the chloride or sulfate form; and 3) thereafter conversion of the strong base sites to the carbonate form and the weak base sites to the free base form. The plant protein containing phytate is then contacted with the treated resin to remove the phytate. The teachings of U.S. Pat. No. 5,248,804 are herein incorporated by reference.
Phytate comprises the salts of phytic acid. Phytic acid is also known as inositol hexaphosphate. Thus, in using an anion exchange resin, the highly anionic phosphate groups provide the handle by which the resin can extract the phytate from the protein slurry. In contrast, isoflavones are neutral molecules and would not be expected to attach to the resin or exchange with the anions on the resin. The teachings of U.S. Pat. No. 5,248,804 are herein incorporated by reference.
U.S. Pat. No. 5,492,899 to Masor et al. discloses an infant formula with ribo-nucleotides. This patent teaches the use of certain levels and ratios of nucleotide equivalents in infant formulas and discloses an analytical technique to identify and quantify the nucleotide equivalents in a nutritional matrix. The teachings of U.S. Pat. No. 5,392,899 are herein incorporated by reference.
The present invention comprises a method to isolate isoflavones from plant material. The present invention further comprises pharmaceutical products made with the isoflavones produced in accordance with the invention. This, and other aspects of the invention are specifically described in detail in the description set forth below.